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All the mutants except for D150N completely lost their methylation activities.
Interestingly, using COS-1 cells as a eukaryotic expression system, all mutants except for those targeting p.L34 were found to be synthesized and secreted to the cell supernatant, from which they could be purified for further experiments [31],[32].
Soluble sugar levels in leaf sheaths were similar between lse mutants and WT, or were lower in the mutants, except for the fourth leaf sheath of lse2, in which sucrose levels were significantly higher than those of WT in both morning and evening.
Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark.
As shown in Figure 2A, considerable amounts of Swi3 mutant proteins were recovered from all mutants except for swi3-E10.
All xylanase activity assay data correlated well with the thermal unfolding curves of the respective mutants, except for F4A.
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In the dark, one big group, which showed no reaction at all using prolonged times, is composed of L112C-A132C/Basal mutant except for E113C/Basal mutant.
As shown in Table 1, most of the purified-mutants/Basal mutant except for P107C/, N111C/, G114C/, A124C/, L125C/, W126C/, L128C/, and E134C/Basal mutant formed the typical rhodopsin-like chromophore with A280/A500 ratios between 1.7 and 2.5, with an absorption λmax in the visible range varying from 480 nm to 496 nm.
To further examine whether one of more of the 18 remaining genomic changes observed in the HI mutant (except for the hit locus) are linked to the HI phenotype, we randomly selected one of the 6 isolates in which no hit mutation was found.
Typically, 6 L were expressed for each mutant, except for G117E (12 L).
It has been assumed that pyrimethamine resistance conferred by multiple mutations arose through stepwise selection of the S108N single mutant (except for the A16V/S108T allele).
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